Method carboxy terminal protein or peptide sequencing

ABSTRACT

The present invention provides for an efficient and novel method for the C-terminal sequencing of proteins or peptides by use of acetyl chloride or phosphoryl chloride by reaction with a suitable isothiocyanate for derivitazation of the carboxy terminus to a thiohydantoin amino acid derivative, under acidic conditions. Cleavage of the derivatized thiohydantoin amino acid may occur by use of thiocyanic acid and acetic acid in water and also by the novel means using a buffer and a potassium or sodium thiocyanate or potassium or sodium dithionite reagent. The present invention also provides for an novel and efficient means for the C-terminal sequencing of proteins or peptides by a two or three step process which comprises first reacting the peptide or protein with an acid chloride reagent, such as acetyl chloride, or phosphoryl chloride. The stable protein carboxy terminal amino acid chloride is then reacted with an organic isothiocyanate, an organic salt thiocyanate, or a metal thiocyanate to yield a thiohydantoin amino acid derivative. This thiohydantoin amino acid derivative may be cleaved using either acidic or basic condition, preferably using the novel reagents disclosed above.

FIELD OF THE INVENTION

The present invention relates to a method, and reagents useful thereinfor determining the C-terminal amino acid of a peptide or protein andthe process of sequencing the peptide or protein from the C-terminal endthereof.

BACKGROUND OF THE INVENTION

The sequencing of proteins from the carboxyl terminus has been achallenging problem in protein structure determination. Many methods forN-terminal sequencing have been published and readily available to theskilled artisan but little has been accomplished relative to theC-terminus, and fewer methods have resulted in commercial usage.

The thiocyanate method, described by Schlack et al., Physiol. Chem.,154: 125-170 (1926) involved the reaction of a protein or peptide withcertain isothiocyanate reagents, in the presence of acetic anhydride, toform a C-terminal thiohydantoin amino acid. The derivatized amino acidis hydrolyzed to yield a shortened polypeptide and a thiohydantoin aminoacid. The thiohydantoin amino acids are now analyzed by HPLC methods. Adisadvantage of this reaction is the severity of the conditions requiredfor complete derivatization of the C-terminal amino acid.

Stark, G. R., Biochemistry, 7: 1796-1807 (1968) introduced use of thereagent acetohydroxamate as one more mild and capable of performing amore rapid cleavage. U.S. Pat. No. 4,837,165 discloses the use of thereagent trimethylsilylisothiocyanate (TMS-ITC) which resulted in animproved yield of the thiohydantoin formation and reduced the number ofside products obtained. The yields were low upon repetition whichlimited the number of degradation cycles to be performed. Further, notall amino acids were able to form thiohydantoin derivatives by thismethod.

Bailey et al., U.S. Pat. No. 5,180,807; discloses the use as asequencing agent of an isothiocyanate regent which is a combination ofphosphoroisothiocyanatidate and pyridine. The phophoroisothiocyanatidateincludes a diphenyl moiety of the formula (PhO)₂ --P(O)--NCS or adiethyl derivative (EtO)₂ --P(O)--NCS. Bailey et al, U.S. Pat. No.5,227,309 discloses using an alkyl or aryl tin isothiocyanatederivative, such as R_(x) Sn(NCS)_(y). Barley et al., U.S. Pat. No.5,254,475 discloses use of alkali metal salts of trialkyl silanols andtrialkylamine N-oxides, utilized in preference with silyl isothiocyanateas the coupling reagent.

Boyd et al., U.S. Pat. No. 5,185,266 discloses a method for cleaving theacyl thiohydantoin bond with an alkylating agent to form an adduct onthe thiohydantoin. The adduct containing acyl-thiohydantoin is cleavedby reaction under substantially anhydrous acidic conditions.

Boyd et al., U.S. Pat. No. 5,051,368 discloses a method for formingthiohydantoins by activation of the amino acid with a ketenimine, andconverting the ester to a thiohydantoin by reaction with a silyl orpyridine isothiocyanate.

Boyd et al., U.S. Pat. No. 5,041,388 discloses use of a mixed anhydrideof isothiocyanic acid and a carboxylic or carbonic acid, for use underbasic condition which reacts the peptide with the activated supportderivatized as noted.

Boyd et al., U.S. Pat. No. 5,304,497 discloses a method of forming anN-protected amino acid for use in C-terminal peptide sequencing whichutilizes uronium compounds with preferred thiocyanate, such as TMS-ITC,or crown ether adducts of metallothiocyanates.

Miller, U.S. Pat. No. 4,935,494 discloses phophoryl (thio)amide couplingreagents to yield arylthiohydantoin derivatives of amino acids. Milleret al., U.S. Pat. No. 5,066,785 discloses a coupling reagent for use inC-terminal peptides of the formula (R₁)_(n) X_(a) --P(═X_(c))((R₂)_(n)X_(b))N(R₃)--C(═X_(d))--X_(e).

Patent Applications to Hawke et al., U.S. Pat. No. 5,049,507 discloses amethod of C-terminal sequencing wherein the peptide is reacted with amixed anhydride of isothiocyanic acid and a carboxylic, carbonic orsulfonic acid under basic conditions.

Various substrate materials for use in sequencing C-terminal peptideshave been proposed by Bailey et al., in U.S. Pat. No. 5,306,781 whichdiscloses an activated carboxylic acid modified polyethylene membraneand in Sherrington et al., in U.S. Pat. No. 5,066,784 which discloses aporous polymeric material.

SUMMARY OF THE INVENTION

The present invention is to the preparation of thiohydantoin amino acids(TH-AAs) at the C-terminal of proteins by use of the reagentacetylisothiocyanate (Ac-NCS) under acidic conditions in a one stepprocess suitable for manual sequencing. The thiocyanate mediatedcleavage (acidic or basic) of the TH-AAs from proteins constitutes anovel chemistry for C-terminal sequencing of proteins and is comparableto the known Edman degradation of proteins and peptides.

Another embodiment of the present invention is the preparation ofthiohydantoin amino acids (TH-AAs) at the C-terminal of proteins by useof an acid chloride, preferably the reagent acetyl chloride, or tocreate an protein carboxy chloride derivative which is then reacted witha suitable isothiocyanate to form the thiohydantoin amino acid (TH-AAs)which may be cleaved under acidic or basic conditions. This two stepreaction process is appropriately suited to use in a commercial setting,such as in an automated sequencing machine.

DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates a proposed reaction scheme for the sequencing anddetermination of a C-terminal amino acids by reaction of a suitableprotein or peptide with the reagent acetylisothiocyanate to form acarboxy-terminal thiohydantoin derivative. The carboxy-terminalthiohydantoin derivative is cleaved in this instance, under basicreaction conditions using a phosphate buffer, and potassium thiocyanateto release the thiohydantoin amino acid derivative which is thenanalyzed by HPLC.

FIG. 2 demonstrates a proposed reaction scheme for the sequencing anddetermination of a C-terminal amino acids by reaction of an immobilizedsuitable protein or peptide with the reagent acetylisothiocyanate toform a carboxy-terminal thiohydantoin derivative. The carboxy-terminalthiohydantoin derivative is cleaved in this instance, using thiocyanicacid and acetic acid

FIG. 3 demonstrates a graph which shows the stability of the C-terminalthiohydantoin amino acids over a pH range. At elevated temperatures orunder increasing pH the thiohydantoin derivative is not stable andcleaves from the carboxy end of the protein or peptide.

FIG. 4 demonstrates a temperature dependency, as analyzed by HPLC, onthe formation of the thiohydantoin amino acid derivative of the carboxyterminus.

FIG. 5 demonstrates a time dependency, as analyzed by HPLC, on theformation of the thiohydantoin amino acid derivative of the carboxyterminus.

FIG. 6 demonstrates an absorption spectra of the thiohydantoin aminoacid derivatives of proline and threonine prepared in mg amounts.

FIG. 7 demonstrates an analysis of a mixture of amino acid thiohydantoinstandards as analyzed by HPLC. The large peak present in the amino acidsis presumably due to the acetyl-SCN formed along with the acetyl-NCS andis easily removed by the treatment, for instance, with base.

FIG. 8 demonstrates the C-terminal sequencing of the protein lysozyme byHPLC analysis for three cycles.

FIG. 9 demonstrates the C-terminal sequencing of a mixture of threepeptides, GGI, GGL, GGF by HPLC analysis for one cycle.

FIG. 10 demonstrates a proposed reaction scheme for the sequencing anddetermination of a C-terminal amino acids by reaction of an immobilizedsuitable protein or peptide with an acid chlorides and thiocyanate saltsto form a carboxy terminal thiohydantoin derivative. Thecarboxy-terminal thiohydantoin derivative is cleaved in this instance,under basic reaction conditions using a phosphate buffer, and potassiumthiocyanate to release the thiohydantoin amino acid derivative which isthen analyzed by HPLC.

FIG. 11 demonstrates the C-terminal sequencing of a mixture of threepeptides, GGI, GGL, GGF by HPLC analysis of their thiohydantoinformation using three acid chlorides and potassium thiocyanate.

DETAILED DESCRIPTION OF THE INVENTION

There are many problems in carboxy-terminal sequencing which includechemistry of C-terminal derivatization; cleavage of the C-terminalderivatives; identification of the derivatives; availability of properamino acid standards; and the yield of thiohydantoins at each step. Theinstant invention provides for readily accessible chemistry to yield aderivatized thiohydantoin derivative. The thiohydantoin derivative isreadily cleavable, in good yield, to allow identification of theresultant amino acid using known techniques and standards.

One aspect of the present invention is the discovery of a C-terminalderivatization process which yields thiohydantoin amino acids (TH-AAs)through use of acetylchloride (Ac-Cl) and a suitablethiocynate/isothiocyanate to yield the reagent acetyl isothiocyanate(Ac-NCS); or the use of phosphoryl chloride P(O)Cl₃ and a suitablethiocyanate/isothiocyanate to yield reagent P(O)Cl₂ --NCS(P(O)NCS) formanual sequencing. Preferably the final reagent used is Ac-NCS.

It is recognized that reaction of the P(O)Cl₃ moiety with anisothiocyanate will yield combinations of final reagents, ofP(O)Cl(NCS)₂, P(O)Cl₂ NCS or P(O)(NCS)₃ or combinations thereof. All areacceptable for use herein, their amounts determined by molarequivalents, as one skilled in the art would readily recognize.

One embodiment, therefore, of the present invention, is the preparationof thiohydantoin amino acids (TH-AAs) at the C-terminal of proteins byuse of the reagents Ac-NCS, P(O)NCS, or P(Et)NCS, under acidicconditions, in a one step process, which process is suitable for manualsequencing. The thiocyanate mediated cleavage (acidic or basic) of theTH-AAs from proteins constitutes novel chemistry for C-terminalsequencing of proteins and is comparable to the known Edman degradationof proteins and peptides.

An advantage of this invention is that the acetyl isothiocyanate can bereadily prepared in short time, approximately 10 minutes, and thethiohydantoin standards are also easily prepared from free amino acids.

Another aspect of the present invention is the novel cleavage of theTH-AA's from the peptide, or free amino acids for use in makingstandards. As described herein, cleavage of the C-terminal derivatizedTH-AAs from the protein may be carried out under either acidic or basicconditions. A preferred embodiment of the present invention is the useof the novel reagent, a mixture a thiocyanate, preferably an alkalimetal or alkaline earth metal thiocyanate, more preferably sodium orpotassium thiocyanate, or an alkali metal or alkaline earth metaldi-thionite (S₂ O₄), preferably potassium or sodium di-thionite (S₂ O₄),and a buffer, such as a Na phosphate buffer, carbonate buffer or aborate buffer, preferably a Na phosphate buffer, in an organic solvent.Suitably, the molar concentration of each is about 0.01M to 0.2M,preferably about 0.1M. Suitably the organic solvent is acetonitrile,short chain alcohol, such as methanol, isopropanol, ethanol, t-butanol,n-butanol, preferably acetonitrile, from about 0.5-20% (v/v). The pH issuitably from about 8 to 12, suitably from 10 to 12. The reaction cantake place at ambient temperature, or preferably at increasedtemperatures, such as from about 40° to 60° C. The reaction should alsotake from about 5 minutes to several hours, preferably from about 10 to20 minutes at 60° C. A comparison of room temperature versus elevatedtemperatures for a thiocyanate mediated basic cleavage of the TH-AA fromthe protein is further described in FIG. 3.

Another aspect of the present invention is the novel cleavage of theTH-AA's from the protein under acidic conditions using a suitable acid,such as from about 0.1M to 1.5M thiocyanic acid with acetic acid. Thereaction can take place at ambient temperature or at elevatedtemperatures. The reaction should also take from about 20 minutes to anhours, preferably from about 25 to 45 minutes. A demonstration of acidiccleavage is shown in FIG. 7 which is the final products ofderivatization and cleavage of thiohydantoin amino acids. Free aminoacids were appropriately derivatized with acetylisothiocyanate insolution, to which was added water creating a mixture of thiocyanic acidwith acetic acid in equimolar proportions from use of the Ac-NCS as areagent (for derivatization of the TH-AA).

Another aspect of the present invention is the preparation ofthiohydantoin amino acids (TH-AAs) at the C-terminal of proteins by useof an acid chloride, preferably the reagents acetyl chloride, CH₃ S(O)₂Cl, P(O)Cl₃, diethyl phosphoryl chloride, or phenylene P(O)₃ Cl, tocreate a protein carboxy chloride derivative which is then reacted witha suitable isothiocyanate to form the thiohydantoin amino acid (TH-AAs).This two step reaction process when added to a third, cleavage stepunder acidic or basic conditions, as described above, is appropriatelysuited for use in automated sequencing machines.

As used herein, the peptide or protein moiety is also represented by thefigure: ##STR1##

As used herein, the peptide carboxy chloride derivative is representedby the figure: ##STR2## One Step Process:

In the one step process the isothiocyanate coupling reagents may beprepared with a suitable isothiocyanate reagents, preferably either byreaction with KSCN or with trimethylsilyl isothiocyanate (TMS-ITC).

The C-terminal derivatization is then carried out under acidicconditions, using acetic acid, trifluoroacetic acid and hydrochloricacid, preferably acetic acid. The reaction may be done under temperatureconditions of about 20° to about 80° C., preferably about 65° C.Suitably the time period for reaction is from about 5 minutes to about80 minutes, preferably about 40 min.

Cleavage of the C-terminal TH-AAs from the protein may be carried outunder either acidic or basic conditions as described above. It isrecognized that use of the reagent Ac-NCS in this one step process willproduce acidic conditions for cleavage of the TH-AA's from protein. Suchconditions can be facilitated by treatment of the supported peptide withaddition of water. The reaction can take place at ambient temperature orat any elevated temperature, such from about 40° to 80° C. The reactionshould also take from about 20 minutes to an hours, preferably fromabout 25 to 45 minutes.

Two Step Process:

In a two step process the protein is reacted with a suitable acidchloride derivative, such as acetyl chloride, methyl sulfonyl chloride,or phosphoryl chloride, under acidic conditions to yield a stableprotein carboxy chloride. The protein carboxy chloride is then reacted,under acidic conditions, with either an organic salt thiocyanate, suchas guanidine thiocyanate, or piperidine thiocyanate; an organicisothiocyanate, such TMS-ITC, or a metal thiocyanate, such K+ or Na+(iso)thiocyanate, to form a C-terminal protein thiohydantoin derivative(AA-TH). This reaction is generally described in FIG. 10 and an actualexample is further shown in FIG. 11.

The protein C-terminal derivatization, as noted above, is carried outunder acidic conditions, using a suitable acid, such as acetic acid,trifluoroacetic acid and hydrochloric acid, under varied temperatureconditions from about 20° to 80° C., preferably about 55° to 65° C.Suitably the time period for reaction is from about 5 to 80 minutes,preferably from about 30 to about 45 minutes.

Cleavage of the C-terminal TH-AAs from the protein may be carried outunder either acidic or basic conditions as described above. Suitably forautomated sequencing, the cleavage occurs under basic conditions. Asuitable reagent for such purposes is use of the novel reagents novelreagent, a mixture a thiocyanate, preferably an alkali metal or alkalineearth metal thiocyanate, more preferably sodium or potassiumthiocyanate, or an alkali metal or alkaline earth metal di-thionite (S₂O₄), preferably potassium or sodium di-thionite (S₂ O₄), and a buffer,such as a Na phosphate buffer, carbonate buffer or a borate buffer,preferably a Na phosphate buffer, in an organic solvent. Preferably thereagent is a mixture of a buffer, preferably a Na phosphate buffer, andpotassium or sodium thiocyanate or potassium or sodium di-thionite (S₂O₄) in an organic solvent, such as acetonitrile.

SYNTHETIC EXAMPLES

The invention will now be described by reference to the followingexamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention. All temperatures aregiven in degrees centigrade, all solvents are highest available purityand all reactions run under anhydrous conditions unless otherwiseindicated.

Example 1 Preparation of Acetylisothiocyanate Coupling Solutions

Reagents and Standards: Trimethylsilyliosothiocyanate (TMS-ITC),guanidine thiocyanate (Gu.HSCN) or piperidine HSCN, acetylchloride,acetic anhydride and acetic acid (Aldrich Chemical®).

a) From Gu.SCN: 200 mg of Gu.SCN was dissolved in 0.8 ml of acetonitrilein a screw cap micro centifuge tube (1.6 ml polypropylene freeze vialsfrom Sigma). 50 ul of acetic anhydride and 5 ul of acetic acid wereadded and mixed. To this mixture 0.15 ml of acteylchloride was added andmixed on a Vortex mixer for about 5 minutes. Gu.HCl was precipiated andseparated from the acetylisothiocyanate (Ac-NCS) solution bycentrifugation.

b) From TMS-ITC: To 0.5 ml of acetonitrile, 0.3 ml of TMS-ITC, 0.15 mlof acetyl chloride, 50 ul of acetic anhydride and 5 ul of acetic acidwere added and mixed. In this case there is no need to centrifuge.

Both reagents as produced in steps (a) and (b) produce a similar result.

Example 2 Preparation of Amino Acid Thiohydantoin Standards

Using the acetylisothiocyanate reagent prepared in accordance withExample 1, steps (a) or (b) above the preparation of amino acidthiohydantoin standards is performed using readily available free aminoacids purchased from a commercial source, such as from BeckmanLaboratories or Pierce Chemicals. Free amino acids and small peptidesmay also be purchased from Sigma Chemicals or Aldrich Chemicals.

a) Dry amino acids individually or in a mixture (<10 nmol ea., approx. 3ul of the amino acid standard mixture dried in the screw cap freezevials using an oven or Reacti-Therm heating block at 65° C.) were mixedwith 50-60 ul of the acetylisothiocyanate coupling solution andsonicated for approx. 10 minutes. The vials were then heated at 65° C.for 40 minutes. After cooling the vials to room temperature, thereaction mixture was diluted with water to 0.5 ml and mixed thoroughlyuntil the oily liquid dissolves in water (approx. 5 min). The vials wereheated again at 65° C. for 45-60 minutes. An aliquot of the reactionmixture (25-50 ul) was analyzed by HPLC.

HPLC analysis: Thiohydantoins were separated using a suitable column,such as a Hypercarb pH (3.0×100 mm, graphitized carbon, Shandon fromAlltech) column using 0.1% TFA in water (solvent A) and in acetonitrile(solvent B). Flow rate used was 0.4 ml/min at 35° C. (using a columnheater). A linear gradient of 3 to 60% B at 30 min followed by a washwith 100% B is used for the AA-TH separations. The Trp-TH eluted in thewash. Other suitable columns may be substituted (see Bailey, et al.,Protein Science, 1: 1662-33 (1992)). Thiohydantoins were detected at 265nm except for Ser-Th and Thr-Th which were detected at 319 nm for thedehydrated forms.

The Ser and Thr yield considerable amounts of dehydrated products evenunder these acidic conditions noted herein. Little if any problems havebeen seen with Asp, Pro, S-Cm-Cys and are quite easily made by thisprocedure. The reaction conditions, with suitable modificationsrecognized by one of skill in the art, may be used to prepare 10-100 mgof the A-TH standards rapidly.

For manual sequencing the sequencing yield for the first cycle is >90%,and for the second cycle is about 50% of the first cycle. Presentconditions have Ser and Cys yielding the same thiohydantoin on HPLC.

Example 3 Terminal Carboxy Sequencing of Protein or Peptides

Using the acetylisothiocyanate reagent prepared in accordance withExample 1, steps (a) or (b) above the an immobilized peptide or proteinof choice is prepared for sequencing from the carboxy terminal end. Inthis Example the protein Lysozyme is demonstrated. Alternatively, theproteins recombinant immunoglobulin G (IgG), β-lactoglobulin, and bovineserum albumin (BSA) have been performed using the same procedure asindicated below.

The lysozyme was immobilized on a Sequelon®-DITC (Millipore Corp.Bedford, Mass.) (or equivalent) membrane in accordance withmanufacturer's suggestions. The Sequelon® disk containing the lysozymewas treated with 0.1 to 0.2 ml of acetylisothiocyanate coupling solutionat 45° to 75° C. for, preferably at 65° C, for 40 to 100 min, preferablyfor 40 min. The protein disk was washed successively with acetonitrile,methanol and 30% methanol in water (v/v). The C-terminal thiohydantoinformed was cleaved from the lysozyme using 0.15 ml of 0.1M Na phosphatepH 11 (at 60° C.) or pH 12 (at ambient temperature) containing 0.1M KSCNand 30% methanol solution for 5-80 min, preferably 15 at 60° C. and 40min at room temperature. The protein disk was removed from the cleavingsolution and the thiohydantoin was acidified with dilute acetic acid.The cleaved thiohydantoin was determined by HPLC using a Hypercarbcolumn as in Example 2 above.

For the second cycle the disk containing the protein was washedsuccessively with 5% trifluoroacetic acid in water, 30% methanol-waterand acetonitrile and the derivatization procedure described above wasrepeated in its entirety.

Example 4 Carboxyl-Terminal Derivatization Using Acid Chlorides andThiocyanates

A equimolar peptide mixture of Gly-Gly-Leu, Gly-Gly-Ile and Gly-Gly-Phewas immobilized on a Sequelon®-DITC membrane disk as in Example 3 above.The disk containing the peptide was treated first with excess of acidchlorides (e.g.: acetylchloride, methylsulfonylchloride, phosphorylchloride and diethylphosphorylchloride etc.) at about 55°-60° C., forabout 10 min. The disk was washed with anhydrous acetonitrile and thentreated with thiocyanate solution (e.g.: saturated solution of KSCN inacetone, guanidine thiocyanate in acetonitrile andtrimethylsilyl-isothiocyanate in acetonitrile etc.), preferably about 2Mguanidine thiocyanate in acetonitrile, at 22° C. to 90° C., preferably10 min at 55°-60° C. The disk was washed successively with acetonitrile,methanol and 30% methanol solution (v/v). The thiohydantoins formed werecleaved and determined by HPLC as in Example 2 and 3.

Example 5 Preparation of Amino Acid Thiohydantoin Standards

The following method was used for preparing the amino acid thiohydantoinstandards in large amounts. This procedure is a modification of themethod described in Example 2 although other modifications can be made.4×1.25 g each of guanidine thiocyanate was dissolved in 4×9 ml ofacetonitrile in four separate 15 ml polypropylene screw capped tubes. Toeach tube 0.375 ml of acetic anhydride and 0.065 ml of acetic acid and1.25 ml of acetylchloride were added and vigorously using a shaker(Tekmar). The resulting guanidine.HCl was separated by centrifugationand the supernatant (coupling solution) was combined in a 250 ml roundbottomed flask. About 500 mg of free amino acid was dissolved in thecoupling solution. The reaction mixture was heated at 30° C. to 90° C.,preferably at 60° C., for 20-120 min, preferably for 60 min. Thereaction mixture was cooled to room temperature and diluted with equalvolume of water and re-heated as above, preferably at 60° C. for 60 min.After cooling the reaction mixture was dried using a rotary evaporator.The residue was dissolved in ethyl acetate (˜50-75 ml) and the solublematerial was transferred to an another flask and dried as above. Thisresidue was dissolved in minimum amount warm water and the flask wasleft in a refrigerator overnight. The yellowish powder was separatedfrom the solution, washed with ether or methylene chloride and dried.Yield of the thiohydantoins was depended only on the solubility of theamino acids in the coupling solution. For proline and threonine theyields were ˜90% and 40% respectively. The thiohydantoins were >98% pureby HPLC with a single peak. The elemental analysis and the absorptionspectra were consistent with those of the previously published results.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe are can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

What is claimed is:
 1. A process for sequencing a peptide or protein bycarboxyl terminal derivatization which comprises the reaction of saidpeptide or protein with an isothiocyanate reagent to form acarboxy-terminal thiohydantoin amino acid, wherein the isothiocyanatereagent is selected from acetyl isothiocyanate, P(O)--(Cl)₂ --NCS orP(O)--(Cl)--(NCS)₂.
 2. The process according to claim 1 wherein theisothiocyanate reagent is prepared by reacting acetyl chloride with analkali salt of an isothiocyanate, or an organic isothiocyanate.
 3. Theprocess according to claim 2 wherein the alkali salt of anisothiocyanate is potassium isothiocyanate; and the organicisothiocyanate reagent is trimethylsilyl isothiocyanate.
 4. The processaccording to claim 1 wherein the protein or peptide is covalentlyattached to a solid support.
 5. The process according to claim 1 whereinthe carboxy-terminal thiohydantoin amino acid is cleaved with an acid orbase to release a thiohydantoin amino acid.
 6. The process according toclaim 5, wherein the released thiohydantoin amino acid is analyzed fordetermination of the amino acid.
 7. The process according to claim 5wherein the carboxy-terminal thiohydantoin amino acid cleavage is bythiocyanic acid and acetic acid.
 8. The process according to claim 7wherein the water is added to the reaction mixture.
 9. The processaccording to claim 5 wherein the cleavage is by a mixture of buffer andan alkali metal or alkaline earth metal thiocyanate, or an alkali metalor alkaline earth metal di-thionite (S₂ O₄).
 10. The process accordingto claim 9 wherein the alkali metal or alkaline earth metal thiocyanateis potassium or sodium thiocyanate; and the alkali metal or alkalineearth metal di-thionite (S₂ O₄) is potassium or sodium dithionite. 11.The process according to claim 9 wherein the molar concentration of eachreagent is about 0.01M to 0.2M.
 12. The process according to claim 9which further comprises an organic solvent.
 13. The process according toclaim 12 wherein the organic solvent is acetonitrile.
 14. The processaccording to claim 9 wherein the reaction process is at ambienttemperature, or from about 40° to 60° C.; and the pH is suitably fromabout 8 to
 12. 15. The process according to claim 9 wherein the bufferis a sodium phosphate buffer, carbonate buffer or a borate buffer. 16.The process according to claim 1 wherein the carboxyl terminalderivatization is carried out under acidic conditions, using aceticacid, triflouroacetic acid, hydrochloric acid or mixtures thereof. 17.The process according to claim 1 wherein the isothiocyanate reagent isacetyl isothiocyanate.
 18. The process according to claim 1 wherein theisothiocyanate reagent is P(O)--(Cl)₂ --NCS or P(O)--(Cl)--(NCS)₂.
 19. Aprocess for sequencing a peptide or protein by carboxyl terminalderivatization which comprisesa) reacting said peptide or protein withan isothiocyanate reagent selected from the group consisting of acetylisothiocyanate, P(O)--(Cl)₂ --NCS and P(O)--(Cl)(NCS)₂ forming acarboxy-terminal thiohydantoin amino acid; b) cleaving thecarboxy-terminal thiohydantoin amino acid with an acid or base torelease a thiohydantoin amino acid.
 20. The process according to claim19, wherein the released thiohydantoin amino acid is analyzed fordetermination of the amino acid.